PRENATAL DIAGNOSIS OF β-THALASSEMIA MAJOR BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS OF HEMOGLOBINS IN FETAL BLOOD SAMPLES

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Abstract
In Thailand and adjacent countries, most of the β-thalassemia genes are β0-thalassemia mutations that prevent the production of Hb A. We propose the quantitation of the Hb A fraction in fetal blood in the mid-trimester of pregnancy by automated high performance liquid chromatography as a reasonable prenatal diagnostic method to be applied in areas with limited laboratory facilities. Forty pregnant women at risk of delivering a child with β-thalassemia major were identified using an erythrocyte osmotic fragility test and quantitation of Hb A2. Cordocentesis was performed at the gestational age of 18–22 weeks and fetal blood was analyzed for hemoglobin fractions by automated high performance liquid chromatography. The β-globin gene mutations were characterized by β-globin gene sequencing. The 4 bp deletion at codons 41/42 (−TTCT) was the most frequent of the 40 β-thalassemia mutations observed (20/40 = 50%), followed by the splice site mutation IVS-I-1 (G → T) (7/40 = 17.5%), the nonsense mutation at codon 17 (A → T) (7/40 = 17.5%), the nonsense mutation at codon 35 (C → A) (3/40 = 7.5%), and the β+ -thalassemia promoter mutation at −28 (A → G) (3/40 = 7.5%). High performance liquid chromatography revealed nine fetuses which had only Hb F and no Hb A. All were homozygotes or compound heterozygotes for β0-thalassemia mutations. In the remaining 31 fetuses, a Hb A peak was present in the chromatograms. One fetus with 0.5% Hb A was a compound heterozygote for the −28 (A → G) and codons 41/42 (−TTCT) mutations. In the remaining 30 fetuses, the Hb A values ranged between 0.8 and 7.4%. Twenty of these, with a Hb A concentration of 1.82 ± 0.49% (range 0.8–2.8%), were β-thalassemia heterozygotes. The remaining 10 fetuses had Hb A values of 4.89 ± 1.47% (range 2.9–7.4%) and normal β-globin genes. The absence of Hb A in homozygotes or compound heterozygotes for β0-thalassemia mutations and the presence of measurable amounts of Hb A in heterozygotes and normal homozygotes, permits the diagnosis of fetuses expected to develop postnatal β-thalassemia major.